Novel polypeptide, a cDNA encoding the same, and use of it

ABSTRACT

A new human polypeptide, a cDNA encoding the same and a pharmaceutical use of it.  
     The polypeptides of the present invention possess hematopolesis regulating activity, tissue generation/regeneration activity, activin/inhibin activity, chemotactic/chemokinetic activity, hemostatic and thrombolytic activity, and receptor/ligand activity, therefore, they are expected to be useful for prevention and/or treatment of various diseases.

TECHNICAL FIELD

[0001] The present invention relates to a novel polypeptide, a methodfor producing it, a cDNA encoding it, a vector containing the cDNA, ahost cell transformed with the vector, an antibody against the peptide,and a pharmaceutical composition containing the polypeptide or theantibody.

TECHNICAL BACKGROUND

[0002] Until now, when a man skilled in the art intends to obtain aparticular polypeptide or a cDNA encoding it, he generally utilizesmethods by confirming an aimed biological activity in a tissue or in acell medium, isolating and purifying the polypeptide and then cloning agene or methods by “expression-cloning” with the guidance of the saidbiological activity. However, physiologically active polypeptides inliving body have often many kinds of activities. Therefore, it happensincreasingly that after cloning a gene, the isolated gene is found to beidentical to that encoding a polypeptide already known. In addition,some factors could be generated in only a very slight amount and/orunder specific conditions and it makes difficult to isolate and topurify the factor and to confirm its biological activity.

[0003] Recent rapid developments in techniques for constructing cDNAsand sequencing techniques have made it possible to quickly sequence alarge amount of cDNAs. By utilizing these techniques, a process, whichcomprises constructing cDNAs library using various cells or tissues,cloning the cDNA at random, identifying the nucleotide sequencesthereof, expressing novel polypeptides encoded by them, is now inprogress. Although this process is advantageous in that a gene can becloned and information regarding its nucleotide sequence can be obtainedwithout any biochemical or genetic analysis, the target gene can bediscovered thereby only accidentally in many cases.

DISCLOSURE OF THE INVENTION

[0004] The present inventors have studied cloning method to isolategenes encoding proliferation and/or differentiation factors functioningin hematopoietic systems and immune systems. Focusing their attention onthe fact that most of the secretory proteins such as proliferationand/or differentiation factors (for example, various cytokines) andmembrane proteins such as receptors thereof (hereafter these proteinswill be referred to generally as secretory proteins and the like) havesequences called signal peptides in the N-termini, the inventors haveconducted extensive studies on a process for efficiently and selectivelycloning a gene encoding for a signal peptide. Finally, we havesuccessfully developed a screening method for the signal peptides(signal sequence trap (SST)) by using mammalian cells (See EP-0607054).We also developed yeast SST method on the same concept. By the methodbased on the same conception using yeast, (yeast SST method), genesincluding sequence encoding signal peptide can be identified more easilyand efficiently (See U.S. Pat. No. 5,536,637).

[0005] The present inventors et al. have diligently performed certaininvestigation in order to isolate novel factors (polypeptides) usefulfor treatment, diagnosis and/or study, particularly, secretory proteinscontaining secretory signal and membrane protein. From the result, thepresent inventors achieved to find novel secretory proteins and membraneproteins produced from cell lines and tissue, for example, humanplacenta, human adult brain tissue, cell lines derived from human braintissue, human bone, cell line derived from human bone marrow, andendothelial cell line of vein derived from human umbilical cord(HUV-EC-C) and cDNAs encoding them, and then completed the presentinvention.

[0006] A cDNA nucleotide sequence provided by the present invention wasidentified as clone OAH047. The said clone was isolated from cDNAlibrary synthesized from endothelial cell line of vein derived fromhuman umbilical cord (HUV-EC-C) based on the information obtained byusing the above yeast SST method. Clone OAH047 was full-length cDNAcontaining nucleotide sequence encoding membrane protein (represented asOAH047 protein).

[0007] It was indicated from the results of homology search for thepublic database of the nucleic acid sequences by using BLASTN and FASTA,and for the public database of the amino acid sequences by using BLASTX,BLASTP and FASTA, that there was no sequence identical to thepolypeptide sequence and the nucleotide sequence of OAH047 of thepresent invention. It was also indicated from the hydrophobisityanalysis that the polypeptide of the present invention had notransmembrane region. Taken altogether, it was proved that polypeptideOAH047 of the present invention was new secretary protein.

[0008] That is to say, the invention relates to:

[0009] (1) a polypeptide comprising an amino acid sequence shown in SEQID NO. 1,

[0010] (2) a cDNA encoding the polypeptide described in above (1),

[0011] (3) a cDNA comprising a nucleotide sequence shown in SEQ ID NO.2,

[0012] (4) a cDNA comprising a nucleotide sequence shown in SEQ ID NO.3.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0013] The present invention relates to a polypeptide comprising aminoacid sequence shown in SEQ ID NO. 1 in substantially purified form, ahomologue thereof, a fragment of the sequence and a homologue of thefragment.

[0014] Further, the present invention relates to cDNAs encoding theabove peptides. More particularly the invention is provided cDNAscomprising nucleotide sequence shown in SEQ ID NO. 2 and cDNA containinga fragment which is selectively hybridizing to the cDNA comprisingnucleotide sequences shown in SEQ ID NOS. 2 or 3. A said cDNA capablefor hybridizing to the cDNA includes the contemporary sequence of theabove sequence.

[0015] A polypeptide comprising amino acid sequence shown in SEQ ID NO.1 in substantially purified form will generally comprise the polypeptidein a preparation in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the preparation is that of the SEQ ID NO. 1.

[0016] A homologue of polypeptide comprising amino acid sequence shownin SEQ ID NO. 1 will be generally at least 70%, preferably at least 80or 90% and more preferably at least 95% homologous to the polypeptidecomprising the said amino acid sequence over a region of at least 20,preferably at least 30, for instance 40, 60 or 100 more contiguous aminoacids. Such a polypeptide homologue will be referred to a polypeptide ofthe present invention.

[0017] Generally, a fragment of polypeptide comprising amino acidsequence shown in SEQ ID NO. 1 or its homologues will be at least 10,preferably at least 15, for example, 20, 25, 30, 40, 50 or 60 aminoacids in length.

[0018] A cDNA capable of selectively hybridizing to the cDNA comprisingnucleotide sequences shown in SEQ ID NOS. 2 or 3 will be generally atleast 70%, preferably at least 80 or 90% and more preferably at least95% homologous to the cDNA comprising the said nucleotide sequence overa region of at least 20, preferably at least 30, for instance 40, 60 or100 or more contiguous nucleotides: Such a cDNA will be referred to “acDNA of the present invention”.

[0019] Fragments of the cDNA comprising nucleotide sequences shown inSEQ ID NOS. 2 or 3 will be at least 10, preferably at least 15, forexample, 20, 25, 30 or 40 nucleotides in length, and will be alsoreferred to “a cDNA of the present invention” as used herein.

[0020] A further embodiment of the present invention providesreplication and expression vectors carrying cDNA of the presentinvention. The vectors may be, for example, plasmid, virus or phagevectors provided with an origin of replication, optionally a promoterfor the expression of the said cDNA and optionally a regulator of thepromoter. The vector may contain one or more selectable marker genes,for example, an ampicillin resistance gene. The vector may be used invitro, for example, of the production of RNA corresponding to the cDNA,or used to transfect a host cell.

[0021] A further embodiment of the present invention provides host cellstransformed with the vectors for the replication and expression of thecDNA of the present invention, including the cDNA comprising nucleotidesequences shown in SEQ ID NOS. 2 or 3 or the open reading frame thereof.The cells will be chosen to be compatible with the vector and may forexample, be bacterial, yeast, insect cells or mammalian cells.

[0022] A further embodiment of the present invention provides a methodof producing a polypeptide which comprises culturing host cells of thepresent invention under conditions effective to express a polypeptide ofthe present invention. Preferably, in addition, such a method is carriedout under conditions in which the polypeptide of the present inventionis expressed and then produced from the host cells.

[0023] cDNA of the present invention may also be inserted into thevectors described above in an antisense orientation in order to provefor the production of antisense RNA. Such antisense RNA may be used in amethod of controlling the levels of a polypeptide of the presentinvention in a cell.

[0024] The invention also provides monoclonal or polyclonal antibodiesagainst a polypeptide of the present invention. The invention furtherprovides a process for the production of monoclonal or polyclonalantibodies to the polypeptides of the present invention. Monoclonalantibodies may be prepared by common hybridoma technology usingpolypeptides of the present invention or fragments thereof, as animmunogen. Polyclonal antibodies may also be prepared by common meanswhich comprise inoculating host animals, (for example, a rat or a rabbitetc.), with polypeptides of the present invention and recovering immuneserum.

[0025] The present invention also provides pharmaceutical compositionscontaining a polypeptide of the present invention, or an antibodythereof, in association with a pharmaceutically acceptable diluentand/or carrier.

[0026] The polypeptide of the present invention specified in (1)includes that which a part of their amino acid sequence is lacking(e.g., a polypeptide comprised of the only essential sequence forrevealing a biological activity in an amino acid sequence shown in SEQID NO. 1), that which a part of their amino acid sequence is replaced byother amino acids (e.g., those replaced by an amino acid having asimilar property) and that which other amino acids are added or insertedinto a part of their amino acid sequence, as well as those comprisingthe amino acid sequence shown in SEQ ID NO. 1.

[0027] As known well, there are one to six kinds of codon as thatencoding one amino acid (for example, one kind of codon for Methionine(Met), and six kinds of codon for Leucine (Leu) are known). Accordingly,the nucleotide sequence of cDNA can be changed in order to encode thepolypeptide having the same amino acid sequence.

[0028] The cDNA of the present invention, specified in (2) includes agroup of every nucleotide sequence encoding polypeptides (1) shown inSEQ ID NO. 1. There is a probability that yield of a polypeptide isimproved by changing a nucleotide sequence.

[0029] The cDNA specified in (3) is the embodiment of the cDNA shown in(2), and indicate the sequence of natural form.

[0030] The cDNA shown in (4) indicates the sequence of the cDNAspecified in (3) with natural non-translational region.

[0031] cDNA carrying nucleotide sequence shown in SEQ ID NO. 3 isprepared by the following method:

[0032] Brief description of Yeast SST method (see U.S. Pat. No.5,536,637) is as follows.

[0033] Yeast such as Saccharomyces cerevisiae should secrete invertaseinto the medium in order to take sucrose or raffinose as a source ofenergy or carbon. (Invertase is an enzyme to cleave raffinose intosucrose and melibiose, sucrose into fructose and glucose). It is knownthat many known mammalian signal sequence make yeast secrete itsinvertase. From these knowledge, SST method was developed as a screeningmethod to find novel signal peptide which make it possible can tosecrete yeast invertase from mammalian cDNA library. SST method usesyeast growth on raffinose medium as a marker. Non-secretory typeinvertase gene SUC2 (GENBKNK Accession No. V 01311) lacking initiationcodon ATG was inserted to yeast expression vector to prepare yeast SSTvector pSUC2. In this expression vector, ADH promoter, ADH terminator(both were derived from AAH5 plasmid (Gammerer, Methods in Enzymol. 101,192-201, 1983)), 2μ ori (as a yeast replication origin), TRP1 (as ayeast selective marker), ColE1 ori (as a E. Coli replication origin) andampicillin resistance gene (as a drug resistance marker) were inserted.Mammalian cDNA was inserted into the upstream of SUC2 gene to prepareyeast SST cDNA library. Yeast lacking secretory type invertase, wastransformed with this library. If inserted mammalian cDNA encodes asignal peptide, yeast could survive in raffinose medium as a result ofrestoring secretion of invertase. Only to culture yeast colonies,prepare plasmids and determine the nucleotide sequence of the insertcDNAs, it is possible to identify novel signal peptide rapidly andeasily.

Preparation of Yeast SST cDNA Library is as Follows

[0034] (1) MRNA is isolated from the targeted cells, double-strandsynthesis is performed by using random primer with certain restrictionenzyme (enzyme I) recognition site,

[0035] (2) obtained double-strand cDNA is ligated to adapter containingcertain restriction endonuclease (enzyme II) recognition site, differfrom enzyme I, digested with enzyme I and fractionated in a appropriatesize,

[0036] (3) obtained cDNA fragment is inserted into yeast expressionvector on the upstream region of invertase gene which signal peptide isdeleted and the library was transformed.

Detailed Description of Each Step is as Follows

[0037] In step (1), mRNA is isolated from mammalian organs and celllines stimulate them with appropriate stimulator if necessary) by knownmethods (molecular Cloning (Sambrook, J., Fritsch, E. F. and Maniatis,T., Cold Spring Harbor Laboratory Press, 1989) or Current Protocol inMolecular Biology (F. M. Ausubel et al, John Wiley & Sons, Inc) if notremark especially).

[0038] HUV-EC-C (endothelial cell line of vein derived from humanumbilical cord: ATCC No. CRL-1730) is chosen as a cell line.Double-strand cDNA synthesis using random primer is performed by knownmethods.

[0039] Any sites may be used as restriction endonuclease recognitionsite I which is linked to adapter and restriction endonucleaserecognition site II which is used in step (2), if both sites aredifferent each other. Preferably, XhoI is used as enzyme I and EcoRI asenzyme II.

[0040] In step (2), cDNA is created blunt-ends with T4 DNA polymerase,ligated enzyme II adapter and digested with enzyme I. Fragment cDNA isanalyzed with agarose-gel electrophoresis (AGE) and is selected cDNAfraction ranging in size from 300 to 800 bp. As mentioned above, anyenzyme may be used as enzyme II, if it is not same the enzyme I.

[0041] In step (3), cDNA fragment obtained in step (2) is inserted intoyeast expression vector on the upstream region of invertase gene whichsignal peptide is deleted. E. Coli was transformed with the expressionvector. Many vectors are known as yeast expression plasmid vector. Forexample, YEp24 is also functioned in E. Coli. Preferably, pSUC2 asdescribed above is used.

[0042] Many host E. Coli strains are known for transformation,preferably DH10B competent cell is used. Any known transformation methodis available, preferably it is performed by electropolation method.Transformant is cultured by conventional methods to obtain cDNA libraryfor yeast SST method.

[0043] However not every all of the clones do not contain cDNA fragment.Further all of the gene fragments do not encode unknown signal peptides.It is therefore necessary to screen a gene fragment encoding for anunknown signal peptide from the library.

[0044] That is to say, screening of fragments containing a sequenceencoding an appropriate signal peptide is performed by transformation ofthe cDNA library into Saccharomyces cerevisiae (e. g. YT455 strain)which lack invertase (it may be prepared by known methods).Transformation of yeast is performed by known methods, e. g. lithiumacetate method. Transformant is cultured in a selective medium, thentransferred to a medium containing raffinose as a carbon source.Survival colonies are selected and then prepared plasmid. Survivalcolonies on a raffinose-medium indicates that some signal peptide ofsecretory protein was inserted to this clone.

[0045] As for isolated positive clones, the nucleotide sequence isdetermined. As to a cDNA encodes unknown protein, full-length clone maybe isolated by using cDNA fragment as a probe and then determined toobtain full-length nucleotide sequence. These manipulation is performedby known methods.

[0046] Once the nucleotide sequence shown in SEQ ID NO. 2 is determinedpartially or preferably fully, it is possible to obtain cDNA encodemammalian protein itself, homologue or subset. cDNA library or mRNAderived from mammals was screened by PCR with any synthesizedoligonucleotide primers or by hybridization with any fragment as aprobe. It is possible to obtain cDNA encodes other mammalian homologueprotein from other mammalian cDNA or genome library.

[0047] If a cDNA obtained above contains a nucleotide sequence of cDNAfragment obtained by SST (or consensus sequence thereof), it will bethought that the cDNA-encodes signal peptide. So it is clear that thecDNA will be full-length or almost full (All signal peptides exist atN-termini of a protein and are encoded at 5′-temini of open readingframe of cDNA).

[0048] The confirmation may be carried out by Northern analysis with thesaid cDNA as a probe. It is thought that the cDNA is almost completelength, if length of the cDNA is almost the same length of the mRNAobtained in the hybridizing band.

[0049] Once the nucleotide sequence shown in SEQ ID NO. 2 is determined,cDNAs of the invention are obtained by chemical synthesis, or byhybridization making use of nucleotide fragments which are chemicallysynthesized as a probe. Furthermore, cDNAs of the invention are obtainedin desired amount by transforming a vector that contains the cDNA into aproper host, and culturing the transformant.

[0050] The polypeptides of the present invention may be prepared by:

[0051] (1) isolating and purifying from an organism or a cultured cell,

[0052] (2) chemically synthesizing, or

[0053] (3) using recombinant DNA technology, preferably, by the methoddescribed in (3) in an industrial production.

[0054] Examples of expression system (host-vector system) for producinga polypeptide by using recombinant DNA technology are the expressionsystems of bacteria, yeast, insect cells and mammalian cells.

[0055] In the expression of the polypeptide, for example, in E. Coli,the expression vector is prepared by adding the initiation codon (ATG)to 5′ end of a cDNA encoding mature peptide, connecting the cDNA thusobtained to the downstream of a proper promoter (e. g., trp promoter,lac promoter, λPL promoter, T7 promoter etc.), and then inserting itinto a vector (e. g., pBR322, pUC18, pUC19 etc.) which functions in anE. Coli strain.

[0056] Then, an E. Coli strain (e. g., E. Coli DH1 strain, E. Coli JM109strain, E. Coli HB101 strain, etc.) which is transformed with theexpression vector described above may be cultured in a appropriatemedium to obtain the desired polypeptide. When a signal sequence ofbacteria (e. g., signal sequence of pel B) is utilized, the desiredpolypeptide may be also released in periplasm. Furthermore, a fusionprotein with other polypeptide may be also produced readily.

[0057] In the expression of the polypeptide, for example, in a mammaliancells, for example, the expression vector is prepared by inserting thecDNA encoding nucleotide sequence shown in SEQ ID NO. 3 into thedownstream of a proper promoter (e. g., SV40 promoter, LTR promoter,metallothionein promoter etc.) in a proper vector (e. g., retrovirusvector, papillomavirus vector, vacciniavirus vector, SV40 vector, etc.).A proper mammalian cell (e. g., monkey COS-7 cell, Chinese hamster CHOcell, mouse L cell etc.) is transformed with the expression vector thusobtained, and then the transformant is cultured in a proper medium toexpress the aimed secretory protein and membrane protein of the presentinvention by the following method.

[0058] In case of secretory protein as for the present invention, theaimed polypeptide was expressed in the supernatant of the cells. Inaddition, fusion protein may be prepared by conjugating cDNA fragmentencoding the other polypeptide, for example, Fc portion of antibody.

[0059] On the other hand, in case of membrane protein as for the presentinvention, the aimed polypeptide was expressed on the cell membrane. AcDNA encoding the nucleotide sequence of SEQ ID NO. 2 with deletion ofextracellular region was inserted into the said vector, transfected intothe an adequate mammalian cells to secret the aimed soluble polypeptidein the culture medium. In addition, fusion protein may be prepared byconjugating cDNA fragment encoding the said mutant with deletion ofextracellular region and other polypeptide, for example, Fc portion ofantibody.

[0060] The polypeptide available by the way described above can beisolated and purified by conventional biochemical method.

Industrial Applicability

[0061] It is considered that the polypeptide of the present inventionand a cDNA which encodes the polypeptide will show one or more of theeffects or biological activities (including those which relates to theassays cited below). The effects or biological activities described inrelation to the polypeptide of the present invention are provided byadministration or use of the polypeptide or by administration or use ofa cDNA molecule which encodes the polypeptide (e.g., vector suitable forgene therapy or cDNA introduction).

Cytokine Activity and Cell Proliferation/differentiation Activity

[0062] The protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. Many protein factorsdiscovered to date, including all known cytokines, have exhibitedactivity in one or more factor dependent cell proliferation assays, andhence the assays serve as a convenient confirmation of cytokineactivity. The activity of a polypeptide of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines.

[0063] [Immune stimulating/suppressing Activity]

[0064] The protein of the present invention may also exhibit immunestimulating or immune suppressing activity. The protein of the presentinvention may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by viral infection such as AIDS (HIV) as well as bacterial orfungal infections, or may result from autoimmune disorders. Morespecifically, infectious diseases causes by viral, bacterial, fungal orother infection may be treatable using the polypeptide of the presentinvention, including AIDS (HIV), infections by hepatitis viruses, herpesviruses, mycobacteria, leshmania, malaria and various fungal infectionssuch as candida. Of course, in this regard, the protein of the presentinvention may also be useful where a boost to the immune systemgenerally would be indicated, i.e., in the treatment of cancer.

[0065] The protein of the present invention may be useful in thetreatment of allergic reactions and conditions, such as asthma or otherrespiratory problems. The protein of the present invention may also beuseful in the treatment of the other conditions required to suppress theimmuno system (for example, asthma or respiratory disease.)

[0066] The protein of the present invention may also suppress chronic oracute inflammation, such as, for example, that associated with infectionsuch as septic shock or inflammatory bowel disease such as systemicinflammatory response syndrome (SIRS), Crohn's disease or resulting fromover production of cytokines such as TNF or IL-I wherein the effect wasdemonstrated by IL-11.

[0067] [Hematopoiesis Regulating Activity]

[0068] The protein of the present invention may be useful in regulationof hematopoiesis, and, consequently, in the treatment of myeloid orlymphoid cell deficiencies. Even marginal biological activity in supportof colony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis. The said biological activitiesare concerned with the following all or some example(s). e.g. insupporting the growth and proliferation of erythroid progenitor cellsalone or in combination withother cytokines, thereby indicating utility,for example, in treating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vitro orex-vivo (i.e. in conjunction with bone marrow transplantation) as normalcells or genetically manipulated for gene therapy.

[0069] The activity of the protein of the present invention may, amongother means, be measured by the following methods:

[0070] [Tissue Generation/regeneration Activity]

[0071] The protein of the present invention also may have utility incompositions used for bone, cartilage, tendon, ligament and/or nervetissue growth or regeneration, as well as for wound healing and tissuerepair, and in the treatment of bums, incisions and ulcers.

[0072] The protein of the present invention, which induces cartilageand/or bone growth in circumstances where bone is not normally formed,may be applied to the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing theprotein of the present invention may have prophylactic use in closed aswell as open fracture reduction and also in the improved fixation ofartificial joints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

[0073] The protein of the present invention may also be used in thetreatment of periodontal disease, and in other tooth repair processes.Such agents may provide an environment to attract bone-forming cells,stimulate growth of bone-forming cells or induce differentiation ofprogenitors of bone-forming cells. The protein of the present inventionmay also be useful in the treatment of osteoporosis or osteoarthritis,such as through stimulation of bone and/or cartilage repair or byblocking inflammation or processes of tissue destruction (collagenaseactivity, osteoclast activity, etc.) mediated by inflammatory processes.

[0074] Another category of tissue regeneration activity that may beattributable to the protein of the present invention is tendon/ligamentformation. The protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, may be applied to the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing theprotein inducing a tendon/ligament-like tissue may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide an environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the presentinvention may also be useful in the treatment of tendinitis, Carpaltunnel syndrome and other tendon or ligament defects. The compositionsmay also include an appropriate matrix and/or sequestering agent as acarrier as is well known in the art.

[0075] The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue. i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies. as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, the protein of the present inventionmay be used in the treatment of diseases of the peripheral nervoussystem, such as peripheral nerve injuries, peripheral neuropathy andlocalized neuropathies, and central nervous system diseases, such asAlzheimer's, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis, and Shy-Drager syndrome. Further conditions which maybe treated in accordance with the invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using the polypeptide of the present invention.

[0076] It is expected that the protein of the present invention may alsoexhibit activity for generation of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting theproliferation of cells comprising such tissues. Part of the desiredeffects may be by inhibition of fibrotic scarring to allow normal tissueto regenerate.

[0077] The protein of the present invention may also be useful for gutprotection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage.

[0078] [Activin/Inhibin Activity]

[0079] The protein of the present invention may also exhibit activin- orinhibin-related activities. Inhibins are characterized by their abilityto inhibit the release of follicle stimulating hormone (FSH), whileactivins are characterized by their ability to stimulate the release offollicle stimulating hormone (FSH). Thus, the protein of the presentinvention alone or in heterodimers with a member of the inhibin *afamily, may be useful as a contraceptive based on the ability ofinhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in these mammals. Alternatively,the protein of the present invention, as a homodimer or as a heterodimerwith other protein subunits of the inhibin-*b group, may be useful as afertility inducing therapeutic, based upon the ability of activinmolecules in stimulating FSH release from cells of the anteriorpituitary (See U.S. Pat. No. 4,798,885). The protein of the presentinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

[0080] [Chemotactic/chemokinetic Activity]

[0081] The protein of the present invention may have chemotactic orchemokinetic activity e.g., functioning as a chemokine, for mammaliancells, including, for example, monocytes, neutrophils, T-cells, mastcells, eosinophils and/or endothelial cells. Chemotactic andchemokinetic proteins can be used to mobilize or attract a desired cellpopulation to a desired site of action. Chemotactic or chemokineticproteins provide particular advantages in treatment of wounds and othertrauma to tissues, as well as in treatment of localized infections. Forexample, attraction of lymphocytes, monocytes or neutrophils to tumorsor sites of infection may result in improved immune responses againstthe tumor or infecting agent.

[0082] If a protein or peptide can stimulate, directly or indirectly,the directed orientation or movement of such cell population, it haschemotactic activity for a particular cell population. Preferably, theprotein or peptide has the ability to directly stimulate directedmovement of cells. Whether a particular protein has chemotactic activityfor a population of cells can be readily determined by employing suchprotein or peptide in any known assay for cell chemotaxis.

[0083] [Hemostatic and Thrombolytic Activity]

[0084] The protein of the present invention may also exhibit hemostaticor thrombolyic activity. As a result, such a protein is expected to beuseful in treatment of various coagulation disorders (includinghereditary disorders, such as hemophilias) or to enhance coagulation andother hemostatic events in treating wounds resulting from trauma,surgery or other causes. A protein of the present invention may also beuseful for dissolving or inhibiting formation of thromboses and fortreatment and prevention of conditions resulting therefrom such as, forexample, infarction or stroke.

[0085] [Receptor/ligand Activity]

[0086] The protein of the present invention may also demonstrateactivity as receptors, receptor ligands or inhibitors or agonists ofreceptor/ligand interactions. Examples of such receptors and ligandsinclude, without limitation, cytokine receptors and their ligands,receptor kinases and their ligands, receptor phosphatases and theirligands, receptors involved in cell-cell interactions and their ligands(including cellular adhesion molecules such as Selectins, Integrins andtheir ligands) and receptor/ligand pairs involved in antigenpresentation, antigen recognition and development of cellular andhumoral immune responses. Receptors and ligands are also useful forscreening of potential peptide or small molecule inhibitors of therelevant receptor/ligand interaction. The protein of the presentinvention (including, without limitation, fragments of receptors andligands) may themselves be useful as inhibitors of receptor/ligandinteractions.

[0087] [Other Activity]

[0088] The protein of the present invention may also exhibit one or moreof the following additional activities or effects: inhibiting growth ofor killing the infecting agents including bacteria, viruses, fungi andother parasites; effecting (suppressing or enhancing) bodycharacteristics including height, weight, hair color, eye color, skin,other tissue pigmentation, or organ or body part size (for example,breast augmentation or diminution) etc.; effecting elimination ofdietary fat, protein, carbohydrate; effecting behavioral characteristicsincluding appetite, libido, stress, cognition (including cognitivedisorders), depression and violent behaviors; providing analgesiceffects or other pain reducing effects; promoting differentiation andgrowth of embryonic stem cells in lineages other than hematopoieticlineages; in the case of enzymes, correcting deficiencies of the enzymeand treating deficiency-related diseases.

[0089] The protein with above activities, is suspected to have followingfunctions by itself or interaction with its ligands or receptors orassociation with other molecules. For example, proliferation or celldeath of B cells, T cells and/or mast cells; specific induction bypromotion of class switch of immunoglobulin genes; differentiation of Bcells to antibody-forming cells; proliferation, differentiation, or celldeath of precursors of granulocytes; proliferation, differentiation, orcell death of precursors of monocytes-macrophages; proliferation, of upregulation or cell death of neutrophils, monocytes-macrophages,eosinophils and/or basophils; proliferation, or cell death of precursorsof megakaryocytes; proliferation, differentiation, or cell death ofprecursors of neutrophils; proliferation, differentiation, or cell deathof precursors of T cells and B cells; promotion of production oferythrocytes; sustainment of proliferation of erythrocytes, neutrophils,eosinophils, basophils, monocytes-macrophages, mast cells, precursors ofmegakaryocyte; promotion of migration of neutrophils,monocytes-macrophages, B cells and/or T cells; proliferation or celldeath of thymocytes; suppression of differentiation of adipocytes;proliferation or cell death of natural killer cells; proliferation orcell death of hematopoietic stem cells; suppression of proliferation ofstem cells and each hematopoietic precursor cells; promotion ofdifferentiation from mesenchymal stem cells to osteoblasts orchondrocytes, proliferation or cell death of mesenchymal stem cells,osteoblasts or chondrocytes and promotion of bone absorption byactivation of osteoclasts and promotion of differentiation frommonocytes to osteoclasts.

[0090] The polypeptide of the present invention is also suspected tofunction to nervous system, so expected to have functions below;differentiation to kinds of neurotransmitter-responsive neurons,survival or cell death of these cells; promotion of proliferation orcell death of glial cells; spread of neural dendrites; survival or celldeath of gangriocytes; proliferation, promotion of differentiation, orcell death of astrocytes; proliferation, survival or cell death ofperipheral neurons; proliferation or cell death of Schwann cells;proliferation, survival or cell death of motoneurons.

[0091] Furthermore, in the process of development of early embryonic,the polypeptide of the present invention is expected to promote orinhibit the organogenesis of epidermis, brain, backbone, and nervoussystem by induction of ectoderm, that of notochord connective tissues(bone, muscle, tendon), hemocytes, heart, kidney, and genital organs byinduction of mesoderm, and that of digestive apparatus (stomach,intestine, liver, pancreas), respiratory apparatus (lung, trachea) byinduction of endoderm. In adult, also, this polypeptide is thought toproliferate or inhibit the above organs.

[0092] Therefore, the polypeptide of the present invention itself isexpected to be used as an agent for the prevention or treatment ofdisease of progression or suppression of immune, nervous, or bonemetabolic function, hypoplasia or overgrowth of hematopoietic cells: forexample, inflammatory disease (rheumatism, ulcerative colitis, etc.),decrease of hematopoietic stem cells after bone marrow transplantation,decrease of leukocytes, platelets, B-cells, or T-cells after radiationexposure or chemotherapeutic dosage against cancer or leukemia, anemia,infectious disease, cancer, leukemia, AIDS, bone metabolic disease(osteoporosis etc.), various degenerative disease (Alzheimer's disease,multiple sclerosis, etc.), or nervous lesion.

[0093] In addition, since the polypeptide of the present invention isthought to induce the differentiation or growth of organs derived fromectoderm, mesoderm, and endoderm, this polypeptide is expected to be anagent for tissue repair (epidermis, bone, muscle, tendon, heart, kidney,stomach, intestine, liver, pancreas, lung, and trachea, etc.).

[0094] By using polyclonal or monoclonal antibodies against thepolypeptide of the present invention, quantitation of the saidpolypeptide in the body can be performed. It can be used in the study ofrelationship between this polypeptide and disease or diagnosis ofdisease, and so on. Polyclonal and monoclonal antibodies can be preparedusing this polypeptide or its fragment as an antigen by conventionalmethods.

[0095] Identification, purification or molecular cloning of known orunknown proteins which bind the polypeptide of the present invention(preferably polypeptide of extracellular domain) can be performed usingthe polypeptide of the present invention by, for example, preparation ofthe affinity-column.

[0096] Identification of the downstream signal transmission moleculeswhich interact with the polypeptide of the present invention incytoplasma and molecular cloning of the gene can be performed: bywest-western method using the polypeptide of the present invention(preferably polypeptide of transmembrane region or intracellular domain)or by yeast two-hybrid system using the cDNA (preferably cDNA encodingtransmembrane region or cytoplasmic domain of the polypeptide).

[0097] Agonists/antagonists of this receptor polypeptide and inhibitorsbetween receptor and signal transduction molecules can be screened usingthe polypeptide of the present invention.

[0098] cDNAs of the present invention are useful not only the importantand essential template for the production of the polypeptide of thepresent invention which is expected to be largely useful, but also beuseful for diagnosis or therapy (for example, treatment of gene lacking,treatment to stop the-expression of the polypeptide by antisense DNA(RNA)). Genomic DNA may be isolated with the cDNA of the presentinvention, as a probe. As the same manner, a human gene encoding whichcan be highly homologous to the cDNA of the present invention, that is,which encodes a polypeptide highly homologous to the polypeptide of thepresent invention and a gene of animals excluding mouse which can behighly homologous to the cDNA of the present invention, also may beisolated.

[0099] Application to Medicaments

[0100] The polypeptide of the present invention or the antibody specificfor the polypeptide of the present invention is administeredsystemically or topically and in general orally or parenterally,preferably parenterally, intravenously and intraventricularly, forpreventing or treating the said diseases.

[0101] The doses to be administered depend upon age, body weight,symptom, desired therapeutic effect, route of administration, andduration of the treatment etc. In human adults, one dose per person isgenerally between 100 μg and 100 mg, by oral administration, up toseveral times per day, and between 10 μg and 100 mg, by parentaladministration up to several times per day.

[0102] As mentioned above, the doses to be used depend upon variousconditions. Therefore, there are cases in which doses lower than orgreater than the ranges specified above may be used.

[0103] The compounds of the present invention, may be administered assolid compositions, liquid compositions or other compositions for oraladministration, as injections, liniments or suppositories etc. forparental administration.

[0104] Solid compositions for oral administration include compressedtablets, pills, capsules, dispersible powders, and granules. Capsulesinclude soft or hard capsules.

[0105] In such compositions, one or more of the active compound(s) is orare admixed with at least one inert diluent (such as lactose, mannitol,glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.). Thecompositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (such asmagnesium stearate etc.), disintegrating agents (such as cellulosecalciumglycolate, etc.), stabilizing agents (such as human serumalbumin, lactose etc.), and assisting agents for dissolving (such asarginine, asparaginic acid etc.).

[0106] The tablets or pills may, if desired, be coated with a film ofgastric or enteric materials (such as sugar, gelatin, hydroxypropylcellulose or hydroxypropylmethyl cellulose phthalate, etc.), or becoated with more than two films. And then, coating may includecontainment within capsules of absorbable materials such as gelatin.

[0107] Liquid compositions for oral administration includepharmaceutically acceptable emulsions, solutions, syrups and elixirs. Insuch compositions, one or more of the active compound(s) is or arecontained in inert diluent(s) commonly used (purified water, ethanoletc.). Besides inert diluents, such compositions may also compriseadjuvants (such as wetting agents, suspending agents, etc.), sweeteningagents, flavoring agents, perfuming agents, and preserving agents.

[0108] Other compositions for oral administration include spraycompositions which may be prepared by known methods and which compriseone or more of the active compound(s). Spray compositions may compriseadditional substances other than inert diluents: e.g. stabilizing agents(sodium sulfite etc.), isotonic buffer (sodium chloride, sodium citrate,citric acid, etc.). For preparation of such spray compositions, forexample, the method described in the U.S. Pat. No. 2,868,691 or U.S.Pat. No. 3,095,355 (herein incorporated in their entireties byreference) may be used.

[0109] Injections for parental administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. In such compositions,one or more active compound(s) is or are admixed with at least one inertaqueous diluent(s) (distilled water for injection, physiological saltsolution, etc.) or inert non-aqueous diluents(s)(propylene glycol,polyethylene glycol, olive oil, ethanol, POLYSOLBATE 80 (Trade mark)etc.).

[0110] Injections may comprise additional compound other than inertdiluents: e.g. preserving agents, wetting agents, emulsifying agents,dispersing agents, stabilizing agent (such as human serum albumin,lactose, etc.), and assisting agents such as assisting agents fordissolving (arginine, asparaginic acid, etc.).

BEST MODE CARRYING OUT THE INVENTION

[0111] The invention is illustrated by the following examples relate toclone OAH047, but not limit the invention.

EXAMPLE 1 Preparation of Poly(A)⁺RNA

[0112] Total RNA was extracted from endothelial cell line of veinderived from human umbilical cord (HUV-EC-C) by using TRIzol Reagent(Trade mark, marketed by GIBCOBRL Co.). Poly(A)⁺RNA was purified by mRNAPurification Kit (Trade mark, marketed by Pharmacia).

EXAMPLE 2 Preparation of Yeast SST cDNA Library

[0113] Double strand cDNA was synthesized by Super Script Plasmid systemfor cDNA Synthesis and Plasmid Cloning (Trade name, marketed by GIBCOBRLCo.) with above poly(A)⁺RNA as template and random 9 mer as primer whichwas containing Xhol site:

[0114] 5′-CGATTGAATTCTAGACCTGCCTCGAGNNNNNNNNN-3′ (SEQ ID NO. 4). cDNAwas ligated EcoRI adapter (marketed by GIBCOBRL Co.) by DNA ligation kitver. 2 (Trade name, marketed by Takara-Shuzo Co., this kit was used inall ligating steps hereafter) and digested by XhoI. cDNAs were separatedby agarose-gel electrophoresis. 300-800 bp cDNAs were isolated and wereligated to EcoRI/NotI site of pSUC2 (see U.S. Pat. No. 5,536,637). E.Coli DH10B strains were transformed by PSUC2 with electropolation toobtain yeast SST cDNA library.

EXAMPLE 3 Screening by SST Method and Determination of NucleotideSequence of SST Positive Clone

[0115] Plasmids of the said cDNA library were prepared. Yeast YTK12strains were transformed by the plasmids with lithium acetate method(Current Protocols In Molecular Biology 13.7.1). The transformed yeastwere plated on triptphan-free medium (CMD-Trp medium) for selection. Theplate was incubated for 48 hour at 30° C. Replica of the colony(transformant) which was obtained by Accutran ReplicaPlater (Trade name,marketed by Schleicher & Schuell Co.) were placed onto YPR platecontaining raffinose for carbon source, and the plate was incubated for14 days at 30° C. After 3 days, each colony appeared was streaked on YPRplate again. The plates were incubated for 48 hours at 30° C. Singlecolony was inoculated to YPD medium and was incubated for 48 hours at30° C. Then plasmids were prepared. Insert cDNA was amplified by PCRwith two kind primers which exist end side of cloning site on pSUC2(sense strand primers were biotinylated). Biotinylated single strand ofcDNAs were purified with Dynabeads (Trade name, marketed by DYNAL Co.)and the nucleotide sequences were determined. Sequencing was performedby Dye Terminator Cycle Sequencing Ready Reaction with DNA Sequencingkit (Trade name, marketed by Applied Biosystems Inc.) and sequence wasdetermined by DNA sequencer 373 (Applied Biosystems Inc.) (Allsequencing hereafter was carried out with this method).

[0116] We tried to carry out cloning of full-length cDNA which wasproved to be new one according to the homology search for the obtainednucleotide sequences and deduced amino acid sequences in data base.

EXAMPLE 4 Cloning of a Full-length cDNA and Determination of NucleotideSequence

[0117] A full-length cDNA was cloned using Marathon cDNA AmplificationKit (Trade name, marketed by Clontech Co.) according to 3′RACE (RapidAmplification of cDNA End) method. Double strands cDNA was prepared frompoly(A)⁺RNA in each clone, i.e., endothelial cell line of vein derivedfrom human umbilical cord. 27 mer primer OAH047-F1:

[0118] 5′-GCGACACGTGGATCCAAGATGGCGACG-3′ (SEQ ID NO. 5) containing thededuced initiation ATG codon region based on the information ofnucleotide sequence obtained by SST, was prepared. PCR was performedwith the said primer and adapter primer attached in the kit. A cDNAwhich was amplified with clone OAH047 specifically, was separated withagarose-gel electrophoresis, ligated to pT7 Blue-2 T-Vector (Trade name,marketed by Novagen Co.) and transfected into E. Coli DH5a to preparethe plasmid. Nucleotide sequences of 5′-end were determined, and theexistence of nucleotide sequence OAH047 SST cDNA was confirmed.Nucleotide sequence of full-length OAH047 SST cDNA was determined andthen sequence shown in SEQ ID NO. 3 was obtained. An open reading framewas determined and deduced amino acid sequence and nucleotide sequenceshown in SEQ ID NOS. 1 and 2, respectively, were obtained.

[0119] It was indicated from the results of homology search for thepublic database of the nucleic acid sequences by using BLASTN and FASTA,and for the public database of the amino acid sequences by using BLASTX,BLASTP and FASTA, that there was no sequence identical to thepolypeptide sequence and the nucleotide sequence of OAH047 of thepresent invention. In addition, the polypeptide of the present inventionwas expected to possess the transmembrane region by hydrophobisityanalysis of the obtained amino acid sequence. From these results, it wasproved that polypeptide of the present invention was new membraneprotein. Further, the search using BLASTX, BLASTP and FASTA revealed asignificant homology between clone OAH047 (region of 12th -642nd aminoacid in SEQ ID NO. 1) and yeast endomembrane protein (Genbank AccessionU53880, region of 11th -667th amino acid). Based on these homologies,clone OAH047 was expected to functionate transportation of hormone orthe corresponding molecular from cell membrane to lysosome as atranspotor.

1 5 1 642 PRT Homo sapiens mat_peptide (24)..() 1 Met Ala Thr Ala MetAsp Trp Leu Pro Trp Ser Leu Leu Leu Phe Ser -20 -15 -10 Leu Met Cys GluThr Ser Ala Phe Tyr Val Pro Gly Val Ala Pro Ile -5 -1 1 5 Asn Phe HisGln Asn Asp Pro Val Glu Ile Lys Ala Val Lys Leu Thr 10 15 20 25 Ser SerArg Thr Gln Leu Pro Tyr Glu Tyr Tyr Ser Leu Pro Phe Cys 30 35 40 Gln ProSer Lys Ile Thr Tyr Lys Ala Glu Asn Leu Gly Glu Val Leu 45 50 55 Arg GlyAsp Arg Ile Val Asn Thr Pro Phe Gln Val Leu Met Asn Ser 60 65 70 Glu LysLys Cys Glu Val Leu Cys Ser Gln Ser Asn Lys Pro Val Thr 75 80 85 Leu ThrVal Glu Gln Ser Arg Leu Val Ala Glu Arg Ile Thr Glu Asp 90 95 100 105Tyr Tyr Val His Leu Ile Ala Asp Asn Leu Pro Val Ala Thr Arg Leu 110 115120 Glu Leu Tyr Ser Asn Arg Asp Ser Asp Asp Lys Lys Lys Glu Lys Asp 125130 135 Val Gln Phe Glu His Gly Tyr Arg Leu Gly Phe Thr Asp Val Asn Lys140 145 150 Ile Tyr Leu His Asn His Leu Ser Phe Ile Leu Tyr Tyr His ArgGlu 155 160 165 Asp Met Glu Glu Asp Gln Glu His Thr Tyr Arg Val Val ArgPhe Glu 170 175 180 185 Val Ile Pro Gln Ser Ile Arg Leu Glu Asp Leu LysAla Asp Glu Lys 190 195 200 Ser Ser Cys Thr Leu Pro Glu Gly Thr Asn SerSer Pro Gln Glu Ile 205 210 215 Asp Pro Thr Lys Glu Asn Gln Leu Tyr PheThr Tyr Ser Val His Trp 220 225 230 Glu Glu Ser Asp Ile Lys Trp Ala SerArg Trp Asp Thr Tyr Leu Thr 235 240 245 Met Ser Asp Val Gln Ile His TrpPhe Ser Ile Ile Asn Ser Val Val 250 255 260 265 Val Val Phe Phe Leu SerGly Ile Leu Ser Met Ile Ile Ile Arg Thr 270 275 280 Leu Arg Lys Asp IleAla Asn Tyr Asn Lys Glu Asp Asp Ile Glu Asp 285 290 295 Thr Met Glu GluSer Gly Trp Lys Leu Val His Gly Asp Val Phe Arg 300 305 310 Pro Pro GlnTyr Pro Met Ile Leu Ser Ser Leu Leu Gly Ser Gly Ile 315 320 325 Gln LeuPhe Cys Met Ile Leu Ile Val Ile Phe Val Ala Met Leu Gly 330 335 340 345Met Leu Ser Pro Ser Ser Arg Gly Ala Leu Met Thr Thr Ala Cys Phe 350 355360 Leu Phe Met Phe Met Gly Val Phe Gly Gly Phe Ser Ala Gly Arg Leu 365370 375 Tyr Arg Thr Leu Lys Gly His Arg Trp Lys Lys Gly Ala Phe Cys Thr380 385 390 Ala Thr Leu Tyr Pro Gly Val Val Phe Gly Ile Cys Phe Val LeuAsn 395 400 405 Cys Phe Ile Trp Gly Lys His Ser Ser Gly Ala Val Pro PhePro Thr 410 415 420 425 Met Val Ala Leu Leu Cys Met Trp Phe Gly Ile SerLeu Pro Leu Val 430 435 440 Tyr Leu Gly Tyr Tyr Phe Gly Phe Arg Lys GlnPro Tyr Asp Asn Pro 445 450 455 Val Arg Thr Asn Gln Ile Pro Arg Gln IlePro Glu Gln Arg Trp Tyr 460 465 470 Met Asn Arg Phe Val Gly Ile Leu MetAla Gly Ile Leu Pro Phe Gly 475 480 485 Ala Met Phe Ile Glu Leu Phe PheIle Phe Ser Ala Ile Trp Glu Asn 490 495 500 505 Gln Phe Tyr Tyr Leu PheGly Phe Leu Phe Leu Val Phe Ile Ile Leu 510 515 520 Val Val Ser Cys SerGln Ile Ser Ile Val Met Val Tyr Phe Gln Leu 525 530 535 Cys Ala Glu AspTyr Arg Trp Trp Trp Arg Asn Phe Leu Val Ser Gly 540 545 550 Gly Ser AlaPhe Tyr Val Leu Val Tyr Ala Ile Phe Tyr Phe Val Asn 555 560 565 Lys LeuAsp Ile Val Glu Phe Ile Pro Ser Leu Leu Tyr Phe Gly Tyr 570 575 580 585Thr Ala Leu Met Val Leu Ser Phe Trp Leu Leu Thr Gly Thr Ile Gly 590 595600 Phe Tyr Ala Ala Tyr Met Phe Val Arg Lys Ile Tyr Ala Ala Val Lys 605610 615 Ile Asp 2 1926 DNA Homo sapiens 2 atggcgacgg cgatggattggttgccgtgg tctttactgc ttttctccct gatgtgtgaa 60 acaagcgcct tctatgtgcctggggtcgcg cctatcaact tccaccagaa cgatcccgta 120 gaaatcaagg ctgtgaagctcaccagctct cgaacccagc taccttatga atactattca 180 ctgcccttct gccagcccagcaagataacc tacaaggcag agaatctggg agaggtgctg 240 agaggggacc ggattgtcaacacccctttc caggttctca tgaacagcga gaagaagtgt 300 gaagttctgt gcagccagtccaacaagcca gtgaccctga cagtggagca gagccgactc 360 gtggccgagc ggatcacagaagactactac gtccacctca ttgctgacaa cctgcctgtg 420 gccacccggc tggagctctactccaaccga gacagcgatg acaagaagaa ggaaaaagat 480 gtgcagtttg aacacggctaccggctcggc ttcacagatg tcaacaagat ctacctgcac 540 aaccacctct cattcatcctttactatcat cgggaggaca tggaagagga ccaggagcac 600 acgtaccgtg tcgtccgcttcgaggtgatt ccccagagca tcaggctgga ggacctcaaa 660 gcagatgaga agagttcgtgcactctgccc gagggtacca actcctcgcc ccaagaaatt 720 gaccccacca aggagaatcagctgtacttc acctactctg tccactggga ggaaagtgat 780 atcaaatggg cctctcgctgggacacttac ctgaccatga gtgacgtcca gatccactgg 840 ttttctatca ttaactccgttgttgtggtc ttcttcctgt caggtatcct gagcatgatt 900 atcattcgga ccctccggaaggacattgcc aactacaaca aggaggatga cattgaagac 960 accatggagg agtctgggtggaagttggtg cacggcgacg tcttcaggcc cccccagtac 1020 cccatgatcc tcagctccctgctgggctca ggcattcagc tgttctgtat gatcctcatc 1080 gtcatctttg tagccatgcttgggatgctg tcgccctcca gccggggagc tctcatgacc 1140 acagcctgct tcctcttcatgttcatgggg gtgtttggcg gattttctgc tggccgtctg 1200 taccgcactt taaaaggccatcggtggaag aaaggagcct tctgtacggc aactctgtac 1260 cctggtgtgg tttttggcatctgcttcgta ttgaattgct tcatttgggg aaagcactca 1320 tcaggagcgg tgccctttcccaccatggtg gctctgctgt gcatgtggtt cgggatctcc 1380 ctgcccctcg tctacttgggctactacttc ggcttccgaa agcagccata tgacaaccct 1440 gtgcgcacca accagattccccggcagatc cccgagcagc ggtggtacat gaaccgattt 1500 gtgggcatcc tcatggctgggatcttgccc ttcggcgcca tgttcatcga gctcttcttc 1560 atcttcagtg ctatctgggagaatcagttc tattacctct ttggcttcct gttccttgtt 1620 ttcatcatcc tggtggtatcctgttcacaa atcagcatcg tcatggtgta cttccagctg 1680 tgtgcagagg attaccgctggtggtggaga aatttcctag tctccggggg ctctgcattc 1740 tacgtcctgg tttatgccatcttttatttc gttaacaagc tggacatcgt ggagttcatc 1800 ccctctctcc tctactttggctacacggcc ctcatggtct tgtccttctg gctgctaacg 1860 ggtaccatcg gcttctatgcagcctacatg tttgttcgca agatctatgc tgctgtgaag 1920 atagac 1926 3 2083 DNAHomo sapiens misc_feature Clone OAH047 - human umbilical vein HUV-EC-Cendothelial cell 3 gcgacacgtg gatccaag atg gcg acg gcg atg gat tgg ttgccg tgg tct 51 Met Ala Thr Ala Met Asp Trp Leu Pro Trp Ser -20 -15 ttactg ctt ttc tcc ctg atg tgt gaa aca agc gcc ttc tat gtg cct 99 Leu LeuLeu Phe Ser Leu Met Cys Glu Thr Ser Ala Phe Tyr Val Pro -10 -5 -1 1 ggggtc gcg cct atc aac ttc cac cag aac gat ccc gta gaa atc aag 147 Gly ValAla Pro Ile Asn Phe His Gln Asn Asp Pro Val Glu Ile Lys 5 10 15 20 gctgtg aag ctc acc agc tct cga acc cag cta cct tat gaa tac tat 195 Ala ValLys Leu Thr Ser Ser Arg Thr Gln Leu Pro Tyr Glu Tyr Tyr 25 30 35 tca ctgccc ttc tgc cag ccc agc aag ata acc tac aag gca gag aat 243 Ser Leu ProPhe Cys Gln Pro Ser Lys Ile Thr Tyr Lys Ala Glu Asn 40 45 50 ctg gga gaggtg ctg aga ggg gac cgg att gtc aac acc cct ttc cag 291 Leu Gly Glu ValLeu Arg Gly Asp Arg Ile Val Asn Thr Pro Phe Gln 55 60 65 gtt ctc atg aacagc gag aag aag tgt gaa gtt ctg tgc agc cag tcc 339 Val Leu Met Asn SerGlu Lys Lys Cys Glu Val Leu Cys Ser Gln Ser 70 75 80 aac aag cca gtg accctg aca gtg gag cag agc cga ctc gtg gcc gag 387 Asn Lys Pro Val Thr LeuThr Val Glu Gln Ser Arg Leu Val Ala Glu 85 90 95 100 cgg atc aca gaa gactac tac gtc cac ctc att gct gac aac ctg cct 435 Arg Ile Thr Glu Asp TyrTyr Val His Leu Ile Ala Asp Asn Leu Pro 105 110 115 gtg gcc acc cgg ctggag ctc tac tcc aac cga gac agc gat gac aag 483 Val Ala Thr Arg Leu GluLeu Tyr Ser Asn Arg Asp Ser Asp Asp Lys 120 125 130 aag aag gaa aaa gatgtg cag ttt gaa cac ggc tac cgg ctc ggc ttc 531 Lys Lys Glu Lys Asp ValGln Phe Glu His Gly Tyr Arg Leu Gly Phe 135 140 145 aca gat gtc aac aagatc tac ctg cac aac cac ctc tca ttc atc ctt 579 Thr Asp Val Asn Lys IleTyr Leu His Asn His Leu Ser Phe Ile Leu 150 155 160 tac tat cat cgg gaggac atg gaa gag gac cag gag cac acg tac cgt 627 Tyr Tyr His Arg Glu AspMet Glu Glu Asp Gln Glu His Thr Tyr Arg 165 170 175 180 gtc gtc cgc ttcgag gtg att ccc cag agc atc agg ctg gag gac ctc 675 Val Val Arg Phe GluVal Ile Pro Gln Ser Ile Arg Leu Glu Asp Leu 185 190 195 aaa gca gat gagaag agt tcg tgc act ctg ccc gag ggt acc aac tcc 723 Lys Ala Asp Glu LysSer Ser Cys Thr Leu Pro Glu Gly Thr Asn Ser 200 205 210 tcg ccc caa gaaatt gac ccc acc aag gag aat cag ctg tac ttc acc 771 Ser Pro Gln Glu IleAsp Pro Thr Lys Glu Asn Gln Leu Tyr Phe Thr 215 220 225 tac tct gtc cactgg gag gaa agt gat atc aaa tgg gcc tct cgc tgg 819 Tyr Ser Val His TrpGlu Glu Ser Asp Ile Lys Trp Ala Ser Arg Trp 230 235 240 gac act tac ctgacc atg agt gac gtc cag atc cac tgg ttt tct atc 867 Asp Thr Tyr Leu ThrMet Ser Asp Val Gln Ile His Trp Phe Ser Ile 245 250 255 260 att aac tccgtt gtt gtg gtc ttc ttc ctg tca ggt atc ctg agc atg 915 Ile Asn Ser ValVal Val Val Phe Phe Leu Ser Gly Ile Leu Ser Met 265 270 275 att atc attcgg acc ctc cgg aag gac att gcc aac tac aac aag gag 963 Ile Ile Ile ArgThr Leu Arg Lys Asp Ile Ala Asn Tyr Asn Lys Glu 280 285 290 gat gac attgaa gac acc atg gag gag tct ggg tgg aag ttg gtg cac 1011 Asp Asp Ile GluAsp Thr Met Glu Glu Ser Gly Trp Lys Leu Val His 295 300 305 ggc gac gtcttc agg ccc ccc cag tac ccc atg atc ctc agc tcc ctg 1059 Gly Asp Val PheArg Pro Pro Gln Tyr Pro Met Ile Leu Ser Ser Leu 310 315 320 ctg ggc tcaggc att cag ctg ttc tgt atg atc ctc atc gtc atc ttt 1107 Leu Gly Ser GlyIle Gln Leu Phe Cys Met Ile Leu Ile Val Ile Phe 325 330 335 340 gta gccatg ctt ggg atg ctg tcg ccc tcc agc cgg gga gct ctc atg 1155 Val Ala MetLeu Gly Met Leu Ser Pro Ser Ser Arg Gly Ala Leu Met 345 350 355 acc acagcc tgc ttc ctc ttc atg ttc atg ggg gtg ttt ggc gga ttt 1203 Thr Thr AlaCys Phe Leu Phe Met Phe Met Gly Val Phe Gly Gly Phe 360 365 370 tct gctggc cgt ctg tac cgc act tta aaa ggc cat cgg tgg aag aaa 1251 Ser Ala GlyArg Leu Tyr Arg Thr Leu Lys Gly His Arg Trp Lys Lys 375 380 385 gga gccttc tgt acg gca act ctg tac cct ggt gtg gtt ttt ggc atc 1299 Gly Ala PheCys Thr Ala Thr Leu Tyr Pro Gly Val Val Phe Gly Ile 390 395 400 tgc ttcgta ttg aat tgc ttc att tgg gga aag cac tca tca gga gcg 1347 Cys Phe ValLeu Asn Cys Phe Ile Trp Gly Lys His Ser Ser Gly Ala 405 410 415 420 gtgccc ttt ccc acc atg gtg gct ctg ctg tgc atg tgg ttc ggg atc 1395 Val ProPhe Pro Thr Met Val Ala Leu Leu Cys Met Trp Phe Gly Ile 425 430 435 tccctg ccc ctc gtc tac ttg ggc tac tac ttc ggc ttc cga aag cag 1443 Ser LeuPro Leu Val Tyr Leu Gly Tyr Tyr Phe Gly Phe Arg Lys Gln 440 445 450 ccatat gac aac cct gtg cgc acc aac cag att ccc cgg cag atc ccc 1491 Pro TyrAsp Asn Pro Val Arg Thr Asn Gln Ile Pro Arg Gln Ile Pro 455 460 465 gagcag cgg tgg tac atg aac cga ttt gtg ggc atc ctc atg gct ggg 1539 Glu GlnArg Trp Tyr Met Asn Arg Phe Val Gly Ile Leu Met Ala Gly 470 475 480 atcttg ccc ttc ggc gcc atg ttc atc gag ctc ttc ttc atc ttc agt 1587 Ile LeuPro Phe Gly Ala Met Phe Ile Glu Leu Phe Phe Ile Phe Ser 485 490 495 500gct atc tgg gag aat cag ttc tat tac ctc ttt ggc ttc ctg ttc ctt 1635 AlaIle Trp Glu Asn Gln Phe Tyr Tyr Leu Phe Gly Phe Leu Phe Leu 505 510 515gtt ttc atc atc ctg gtg gta tcc tgt tca caa atc agc atc gtc atg 1683 ValPhe Ile Ile Leu Val Val Ser Cys Ser Gln Ile Ser Ile Val Met 520 525 530gtg tac ttc cag ctg tgt gca gag gat tac cgc tgg tgg tgg aga aat 1731 ValTyr Phe Gln Leu Cys Ala Glu Asp Tyr Arg Trp Trp Trp Arg Asn 535 540 545ttc cta gtc tcc ggg ggc tct gca ttc tac gtc ctg gtt tat gcc atc 1779 PheLeu Val Ser Gly Gly Ser Ala Phe Tyr Val Leu Val Tyr Ala Ile 550 555 560ttt tat ttc gtt aac aag ctg gac atc gtg gag ttc atc ccc tct ctc 1827 PheTyr Phe Val Asn Lys Leu Asp Ile Val Glu Phe Ile Pro Ser Leu 565 570 575580 ctc tac ttt ggc tac acg gcc ctc atg gtc ttg tcc ttc tgg ctg cta 1875Leu Tyr Phe Gly Tyr Thr Ala Leu Met Val Leu Ser Phe Trp Leu Leu 585 590595 acg ggt acc atc ggc ttc tat gca gcc tac atg ttt gtt cgc aag atc 1923Thr Gly Thr Ile Gly Phe Tyr Ala Ala Tyr Met Phe Val Arg Lys Ile 600 605610 tat gct gct gtg aag ata gac tgattggagt ggaccacggc caagcctgct 1974Tyr Ala Ala Val Lys Ile Asp 615 ccgtcctcgg acaggaagcc accctgcgtgggggactgcg ggcacgcaaa ataaaataac 2034 tcctgctcgt ttggaatgaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaa 2083 4 35 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 cgattgaatt ctagacctgc ctcgagnnnn nnnnn 35 527 DNA Artificial Sequence Description of Artificial Sequence PrimerOAH047-F1 5 gcgacacgtg gatccaagat ggcgacg 27

1. A substantially purified form of a polypeptide that comprising theamino acid sequence shown in SEQ ID NO. 1, a homologue thereof, afragment thereof or a homologue of the fragment.
 2. A polypeptideaccording to claim 1 that comprising the amino acid sequence shown inSEQ ID NO.
 1. 3. A cDNA encoding the polypeptide according to claim 1.4. A cDNA according to claim 3 that comprising the nucleotide sequenceshown in SEQ ID NO. 2, or a fragment cDNA selectively hybridized to thecDNA.
 5. A cDNA according to claim 3 that comprising the nucleotidesequence shown in SEQ ID NO. 3, or a fragment cDNA selectivelyhybridized to the cDNA.
 6. A replication or expression vector carryingthe cDNA according to claims 3 to
 5. 7. A host cell transformed with thereplication or expression vector according to claim
 6. 8. A method forproducing the polypeptide according to claim 1 or 2 which comprisesculturing a host cell according to claim 7 under a condition effectiveto express the polypeptide according to claim 1 or
 2. 9. A monoclonal orpolyclonal antibody against the polypeptide according to claim 1 or 2.10. A pharmaceutical composition containing the polypeptide according toclaim 1 or 2 or the antibody according to claim 9, in association withpharmaceutically acceptable diluent and/or carrier.